Pathogenic bacterial growth at wound sites, particularly Staphylococcus aureus, poses a serious threat during trauma. Delayed treatment can lead to increased inflammation and severe tissue damage. In this study, a chitosan cross-linked polycationic peptide-conjugated graphene-silver (CGrAP) nanocomposite hydrogel film was developed as an antibacterial wound dressing to treat S. aureus infections. The CGrAP hydrogel was synthesized via a Schiff-base reaction between the ε-poly-L-lysine functionalized graphene-silver nanocomposite and chitosan, and then cast into a film. Its antibacterial action is due to electrostatic interactions and ROS generation, finally disrupting the bacterial cells. In vivo studies on Wistar rat model demonstrated superior bacterial eradication and wound healing compared to antibiotic treatment. The CGrAP hydrogel also showed excellent physicochemical properties, including porosity, water uptake and cytocompatibility with L929 fibroblast cells along with no skin irritation or acute dermal toxicity. These results suggest that, CGrAP nanocomposite hydrogel films have strong potential for antibacterial wound dressing development in chronic wound care.